This invention relates to culturing bone marrow stromal cells.
Bone marrow is a complex and dynamic organ system comprised of hematopoietic cells, bone marrow stromal cells, and extracellular matrix. Pluripotent stem cells within the bone marrow proliferate and differentiate into numerous cell types including erythrocytes and leukocytes. It has been known for some time that association between stem cells and stromal cells is critical for this process. Studies in cell culture have shown that a layer of adherent stromal cells must be established before hematopoietic stem cells can grow and differentiate.
Bone marrow stromal cells are a heterogeneous population of cells that are defined by their morphology and function. In cell culture, they have a characteristic spindle-shaped morphology and secrete growth factors and components that form the extracellular matrix. Stromal cells have been shown to divide in culture in response to epidermal growth factor (EGF; Kimura et al., 1988, Br. J. Hematol. 69:9-12), platelet derived growth factor (PDGF; Kimura et al., supra), and basic fibroblast growth factor (bFGF; Kimura et al., supra; Oliver et al., 1990, Growth Factors, 3:231-236).
The discovery of fibroblast growth factors was based on observations many years ago that extracts from brain could stimulate the division of fibroblasts (see Thomas, 1987, FASEB J., 1:434-440). Particular FGFs have since been isolated and this family now contains at least seven members, including acidic and basic FGF (aFGF and bFGF). These two factors are encoded by different single copy genes and are only 55% identical at the amino acid level (Thomas, supra).
As is the case for most growth factors, FGFs exert their mitogenic activity by binding to cell surface receptors. Currently, four related FGF receptors (FGFR1-FGFR4) have been identified. Each of the FGF receptors exist in multiple forms and the relative affinity of aFGF and bFGF varies for each receptor form (reviewed in Burgess et al., 1989, Ann. Rev. Biochem, 58:575-606; Dionne et al., 1991, Ann. N.Y. Acad. Sci, 638:161-166; Johnson et al., 1993, Adv. Cancer Res. 60:1-41). Basic- and acidic FGF also differ in their response to heparin. In cell culture, heparin complexes with both FGFs, but substantially augments only the mitogenic activity of aFGF (Thomas, supra).
Bone marrow transplantation or implantation is a promising therapy for a number of diseases that involve hematopoietic cells. Transplantation can serve to replace cells that have been damaged by an intrinsic disease, such as an anemia, or in instances where hematopoietic cells have been destroyed by chemotherapy or radiation therapy. Transplantations can be autologous, i.e., the patient can serve as his or her own donor. Alternatively, a patient could receive bone marrow from a histocompatible donor. To date, however, conditions for culturing bone marrow, particularly bone marrow stromal cells, which could be transplanted and used in numerous gene therapies, have not been optimized. A major obstacle to gene therapies that are based on the modification of stromal cells is the procurement of therapeutically useful numbers of stromal cells. Consequently, despite the success of bone marrow transplantation, gene therapies that require successful transplantation of bone marrow stromal cells have not yet been realized.